KIF4A drives gliomas growth by transcriptional repression of Rac1/Cdc42 to induce cytoskeletal remodeling in glioma cells.

Glioma is one of the most prevalent cancers diseases in the worldwide. Kinesin superfamily protein 4 (KIF4), a KIF member classified in Kinesin 4 has been indicated as a mediator acted in tumorigenesis of human cancer. However, the mechanism of KIF4A on glioma is yet to be investigated. This study aimed to explore the potential function and mechanism of KIF4A in gliomas. We analyzed the KIF4A expression and the prognosis in gliomas patients using The Cancer Genome Atlas (TCGA) databases. KIF4A level in normal human astrocyte cell (NHA) and glioma cell lines were examined by Western blot. We studied the function of KIF4A on proliferation, migration, invasion, cell cycle in glioma cell lines using a series of in vitro and in vivo experiments. Chromatin Immunoprecipitation (ChIP) analysis was applied to searching potential KIF4A related downstream in glioma. We identified the significant up-regulated expression of KIF4A both in glioma tissues and cell. Glioma patients with elevated KIF4A expression have shorter survival. Down-regulation of KIF4A exerted inhibitory effect on cell proliferation, invasion and migration. We crucially identified that KIF4A drives gliomas growth by transcriptional repression of Rac1/Cdc42 to induce cytoskeletal remodeling in glioma cells. Knockdown of KIF4A decreased RohA, Rac1, Cdc42, Pak1 and Pak2 expression level. Our study provided a prospect that KIF4A functions as an oncogene in glioma.

Axonal mRNA localization and local translation in neurodegenerative disease.

The regulation of mRNA localization and local translation play vital roles in the maintenance of cellular structure and function. Many human neurodegenerative diseases, such as fragile X syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, and spinal muscular atrophy, have been characterized by pathological changes in neuronal axons, including abnormal mRNA translation, the loss of protein expression, or abnormal axon transport. Moreover, the same protein and mRNA molecules have been associated with variable functions in different diseases due to differences in their interaction networks. In this review, we briefly examine fragile X syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, and spinal muscular atrophy, with a focus on disease pathogenesis with regard to local mRNA translation and axon transport, suggesting possible treatment directions.

Serum soluble tumor necrosis factor-like weak inducer of apoptosis is a potential biomarker for outcome prediction of patients with aneurysmal subarachnoid hemorrhage.

BACKGROUND: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) might contribute to brain inflammation after acute brain injury. The current study was designed to investigate whether serum soluble TWEAK (sTWEAK) can serve as a potential biomarker for functional outcome after aneurysmal subarachnoid hemorrhage (aSAH). METHODS: In this single-center prospective, observational study, admission serum sTWEAK concentrations were quantified among 112 aSAH patients. Impact of serum sTWEAK concentrations on a poor outcome (Glasgow outcome scale score 1-3) at 6 months after stroke onset was determined using multivariate analysis. RESULTS: Admission serum sTWEAK concentrations were intimately correlated with serum C-reactive protein concentrations, World Federation of Neurological Surgeons scores and modified Fisher scores. A total of 38 patients (33.9%) had a poor outcome at post-hemorrhagic 6 months. Admission serum sTWEAK concentrations were substantially higher in patients with a poor outcome than in the other remainders. Under receiver operating characteristic curve, serum sTWEAK concentrations significantly distinguished a poor outcome. Serum sTWEAK concentrations > 3.23 ng/ml discriminated the risk of a poor outcome with medium-high sensitivity and specificity and independently predicted a poor outcome. CONCLUSIONS: Serum sTWEAK, in close correlation with inflammation and hemorrhagic severity, might represent a potential biomarker for predicting clinical outcome after aSAH.

Potential rules of anesthetic gases on glioma.

Glioma is one of the most frequent primary brain tumors. Currently, the most common therapeutic strategy for patients with glioma is surgical resection combined with radiotherapy or/and adjuvant chemotherapy. However, due to the metastatic and invasive nature of glioma cells, the recurrence rate is high, resulting in poor prognosis. In recent years, gas therapy has become an emerging treatment. Studies have shown that the proliferation, metastasis and invasiveness of glioma cells exposed to anesthetic gases are obviously inhibited. Therefore, anesthetic gas may play a special therapeutic role in gliomas. In this review, we aim to collect existing research and summarize the rules of using anesthetic gases on glioma, providing potential strategies for further clinical treatment.

microRNA-200a downregulation in human glioma leads to Gαi1 over-expression, Akt activation, and cell proliferation.

We previously identified a pivotal role for G protein α inhibitory subunit 1 (Gαi1) in mediating PI3K-Akt signaling by receptor tyrosine kinases (RTKs). Here, we examined the expression and biological function of Gαi1 in human glioma. Gαi1 mRNA and protein expression were significantly upregulated in human glioma tissues, which correlated with downregulation of an anti-Gαi1 miRNA: microRNA-200a ("miR-200a"). Forced-expression of miR-200a in established (A172/U251MG lines) and primary (patient-derived) human glioma cells resulted in Gαi1 downregulation, Akt inactivation and proliferation inhibition. Reduction of Gαi1 expression by shRNA, dominant negative mutant interference, or complete Gαi1 depletion inhibited Akt activation and cell proliferation. Notably, miR-200a was unable to inhibit glioma cell proliferation when Gαi1 was silenced or mutated. Co-immunoprecipitation studies, in human glioma cells and tissues, show that Gαi1 forms a complex with multiple RTKs (EGFR, PDGFRα, and FGFR) and the adapter protein Gab1. In vivo, the growth of subcutaneous and orthotopic glioma xenografts in nude mice was largely inhibited by expression of Gαi1 shRNA or miRNA-200a. Collectively, miR-200a downregulation in human glioma leads to Gαi1 over-expression, Akt activation and glioma cell proliferation.

Neuroprotection provided by isoflurane pre-conditioning and post-conditioning.

Isoflurane, a volatile and inhalational anesthetic, has been extensively used in perioperative period for several decades. A large amount of experimental studies have indicated that isoflurane exhibits neuroprotective properties when it is administrated before or after (pre-conditioning and post-conditioning) neurodegenerative diseases (e.g., hypoxic ischemia, stroke and trauma). Multiple mechanisms are involved in isoflurane induced neuroprotection, including activation of glycine and γ-aminobutyric acid receptors, antagonism of ionic channels and alteration of the function and activity of other cellular proteins. Although neuroprotection provided by isoflurane is observed in many animal studies, convincing evidence is lacking in human trials. Therefore, there is still a long way to go before translating its neuroprotective properties into clinical practice.

Hyperbaric oxygen therapy in experimental and clinical stroke.

Stroke, which is defined as a neurologic deficit caused by sudden impaired blood supply, has been considered as a common cause of death and disability for decades. The World Health Organization has declared that almost every 5 seconds a new stroke occurs, placing immense socioeconomic burdens. However, the effective and available treatment strategies are still limited. Additionally, the most effective therapy, such as thrombolysis and stenting for ischemic stroke, generally requires a narrow therapeutic time window after the event. A large majority of patients cannot be admitted to hospital and receive these effective treatments for reperfusion timely. Hyperbaric oxygen therapy (HBOT) has been frequently applied and investigated in stroke since 1960s. Numerous basic and clinical studies have shown the beneficial efficacy for neurological outcome after stroke, and meanwhile many underlying mechanisms associated with neuroprotection have been illustrated, such as cerebral oxygenation promotion and metabolic improvement, blood-brain barrier protection, anti-inflammation and cerebral edema, intracranial pressure modulation, decreased oxidative-stress and apoptosis, increased vascular and neural regeneration. However, HBOT in human stroke is still not sufficiently evidence-based, due to the insufficient randomized double-blind controlled clinical studies. To date, there are no uniform criteria for the dose and session duration of HBOT in different strokes. Furthermore, the additional effect of HBOT combined with drugs and other treatment strategies are being investigated recently. Therefore, more experimental and clinical research is imperative to identify the mechanisms more clearly and to explore the best protocol of HBOT in stroke treatment.

Egr-1 participates in abnormally high gdnf gene transcription mediated by histone hyperacetylation in glioma cells.

Abnormally high transcription of the glial cell-line derived neurotrophic factor (gdnf) gene in glioma cells is related to the hyperacetylation of histone H3 lysine 9 (H3K9) in its promoter region II, but the mechanism remains unclear. There are three consecutive putative binding sites for the transcription factor early growth response protein 1(Egr-1) in promoter region II of the gdnf gene, and Egr-1 participates in gdnf gene transcription activation. Here we show that the acetylation level of H3K9 at Egr-1 binding sites in gdnf gene promoter region II in rat C6 astroglioma cells was significantly higher than that in normal astrocytes, and the binding capacity was also significantly higher. In C6 astroglioma cells, gdnf gene transcription significantly decreased after Egr-1 knock-down. In addition, the deletion or mutation of the Egr-1 binding site also significantly down-regulated the activity of promoter region II of this gene in vitro. When curcumin decreased the acetylation level of H3K9 at the Egr-1 binding site, the binding of Egr-1 to promoter region II and GDNF mRNA levels significantly decreased. In contrast, trichostatin A treatment significantly increased H3K9 acetylation at the Egr-1 binding site, which significantly increased both the binding of Egr-1 with promoter region II and GDNF mRNA levels. In this context, knocking down Egr-1 significantly reduced the elevation in gdnf gene transcription. Collectively, our results demonstrate that the hyperacetylation of H3K9 at Egr-1 binding sites in promoter region II of the gdnf gene can up-regulate the binding of Egr-1 to increase gdnf gene transcription in glioma cells.

Hyperacetylation of histone H3K9 involved in the promotion of abnormally high transcription of the gdnf gene in glioma cells.

The mechanism underlying abnormally high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene in glioma cells is not clear. In this study, to assess histone H3K9 acetylation levels in promoters I and II of the gdnf gene in normal human brain tissue, low- and high-grade glioma tissues, normal rat astrocytes, and rat C6 glioblastoma cells, we employed chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR), real-time PCR, and a pGL3 dual fluorescence reporter system. We also investigated the influence of treatment with curcumin, a histone acetyltransferase inhibitor, and trichostatin A (TSA), a deacetylase inhibitor, on promoter acetylation and activity and messenger RNA (mRNA) expression level of the gdnf gene in C6 cells. Compared to normal brain tissue, H3K9 acetylation in promoters I and II of the gdnf gene increased significantly in high-grade glioma tissues but not in low-grade glioma tissues. Moreover, H3K9 promoter acetylation level of the gdnf gene in C6 cells was also remarkably higher than in normal astrocytes. In C6 cells, curcumin markedly decreased promoter II acetylation and activity and GDNF mRNA expression. Conversely, all three measurements were significantly increased following TSA treatment. Our results suggest that histone H3K9 hyperacetylation in promoter II of the gdnf gene might be one of the reasons for its abnormal high transcription in glioma cells.

The short chain cell-permeable ceramide (C6) restores cell apoptosis and perifosine sensitivity in cultured glioblastoma cells.

Primary glioblastoma multiforme is the most malignant form of astrocytic tumor with an average survival of approximately 12-14 months. The combination of novel Akt inhibitors with anti-cancer therapeutics has achieved improved anti-tumor efficiency. In the current study, we examined the synergistic anti-cancer ability of Akt inhibitor perifosine in combination with short-chain ceramide (C6) against glioblastoma cells (U87MG and U251MG), and studied the underlying mechanisms. We found that perifosine, which blocked Akt/mammalian target of rapamycin activation, only induced moderate cell death and few cell apoptosis in cultured glioblastoma cells. On the other hand, perifosine administration induced significant protective autophagy, which inhibited cell apoptosis induction. Inhibition of autophagy by 3-methyaldenine or by autophagy-related gene-5 RNA interference significantly enhanced perifosine-induced apoptosis and cytotoxicity. We found that the short chain cell-permeable ceramide (C6) significantly enhanced cytotoxic effects of perifosine in cultured glioblastoma cells. For mechanism study, we observed that ceramide (C6) inhibited autophagy induction to restore cell apoptosis and perifosine sensitivity. In conclusion, our study suggests that autophagy inhibition by ceramide (C6) restores perifosine-induced apoptosis and cytotoxicity in glioblastoma cells.

Intraoperative microvascular Doppler monitoring in intracranial aneurysm surgery.

BACKGROUND: Surgical treatment of intracranial aneurysms is often compromised by incomplete exclusion of the aneurysm or stenosis of parent vessels. Intraoperative microvascular Doppler (IMD) is an attractive, noninvasive, and inexpensive tool. The present study aimed to evaluate the usefulness and reliability of IMD for guiding clip placement in aneurysm surgery. METHODS: A total of 92 patients with 101 intracranial aneurysms were included in the study. IMD with a 1.5-mm diameter, 20-MHz microprobe was used before and after clip application to confirm aneurysm obliteration and patency of parent vessels and branching arteries. IMD findings were verified postoperatively with digital subtraction angiography (DSA) or dual energy computed tomography angiography (DE-CTA). Ninety consecutive patients, harboring 108 aneurysms, who underwent surgery without IMD was considered as the control group. RESULTS: The microprobe detected all vessels of the Circle of Willis and their major branches. Clips were repositioned in 24 (23.8%) aneurysms on the basis of the IMD findings consistent with incomplete exclusion and/or stenosis. IMD identified persistent weak blood flow through the aneurismal sac of 11 of the 101 (10.9%) aneurysms requiring clip adjustment. Stenosis or occlusion of the parent or branching arteries as indicated by IMD necessitated immediate clip adjustment in 19 aneurysms (18.8%). The mean duration of the IMD procedure was 4.8 minutes. The frequency of clip adjustment (mean: 1.8 times per case) was associated with the size and location of the aneurysm. There were no complications related to the use of IMD, and postoperative angiograms confirmed complete aneurysm exclusion and parent vessel patency. About 8.3% (9/108) aneurysms were unexpectedly incompletely occluded, and 10.2% (11/108) aneurysms and parent vessel stenosis without IMD were detected by postoperative DSA or DE-CTA. IMD could reduce the rate of residual aneurysm and unanticipated vessel stenosis which demonstrated statistically significant advantages compared with aneurysm surgery without IMD. CONCLUSION: IMD is a safe, easily performed, reliable, and valuable tool that is suitable for routine use in intracranial surgery, especially in complicated, large, and giant aneurysms with wide neck or without neck.

Changes in transcriptional factor binding capacity resulting from promoter region methylation induce aberrantly high GDNF expression in human glioma.

Glial cell line-derived neurotrophic factor (GDNF), which belongs to transforming growth factor ß superfamily, plays important roles in glioma pathogenesis. Gdnf mRNA is aberrantly increased in glioma cells, but the underlying transcription mechanism is unclear. Here, we found that although the base sequence in the promoter region of the gdnf gene was unchanged in glioma cells, there were significant changes in the methylation level of promoter region I (P < 0.05) in both high- and low-grade glioma tissues. However, the methylation degree in promoter region II was notably decreased in low-grade glioma tissue compared to normal brain tissue (P < 0.05), and the demethylation sites were mainly located in the enhancer region. Conversely, methylation was markedly increased in high-grade glioma tissue (P < 0.05), and the sites with decreased methylation level were mainly located in the silencer region. The binding capacities of several transcriptional factors, such as activating protein 2, specificity protein 1, ETS-related gene 2, and cAMP response element binding protein, which specifically bind to regions with altered methylation status decreased along with the pathological grade of glioma, and the differences between high-grade glioma and normal brain tissue were significant (P < 0.05). The results suggest that changes in transcriptional factor binding capacity are due to changes in promoter region methylation and might be the underlying mechanism for aberrantly high gdnf expression in glioma.

BRG1 expression is increased in human glioma and controls glioma cell proliferation, migration and invasion in vitro.

PURPOSE: The purposes of our study were to elucidate the role of BRG1 in the development of human glioma and to determine the effect of BRG1 on glioma cell growth, migration and invasion. METHODS: Using tissue microarray and immunohistochemistry, we evaluated BRG1 staining in 190 glioma tissues, 8 normal brain tissues and 8 tumor adjacent normal brain tissues. We studied glioma cell proliferative ability with reduced BRG1 expression by siRNA using CCK-8 cell proliferation assay and cell cycle analysis. We studied the role of BRG1 in glioma cell migration and invasion by cell migration assay and matrigel invasion assay. We performed western blot to detect cyclin D1, cyclin B1 and MMP-2 protein expression. We also detected MMP-2 enzyme activity by gelatin zymography. RESULTS: Our results showed that BRG1 expression was increased in benign tumor and malignant tumor compared with tumor adjacent normal brain tissue (P < 0.01 for both). We did not find any correlation between BRG1 expression and clinicopathological parameters. In addition, we found that knockdown of BRG1 in glioma cell lines inhibits cell growth due to the G1 phase arrest by downregulating cyclin D1. We further demonstrated that silencing of BRG1 in glioma cells inhibited the cell migration and invasion abilities, and downregulation of MMP-2 expression greatly contributed to the reduced cell invasion and migration abilities. CONCLUSIONS: Our data indicated that BRG1 expression is significantly increased in human glioma and it may be involved in the process of glioma cell proliferation, migration and invasion.

Genetic ablation of toll-like receptor 2 reduces secondary brain injury caused by cortical contusion in mice.

Previous studies have shown that Toll-like receptor 2 (TLR2) was up-regulated after traumatic brain injury (TBI), but the potential contribution of TLR2 to TBI still remains unclear. The present study investigated the role of TLR2 in modulating TBI-induced secondary brain injury in mice. Wild-type TLR2(+/+) and TLR2(-/-)-deficient mice were subjected to a moderately severe weight-drop impact head injury. Brain samples were extracted at 24 hours after trauma. We measured TLR2 by western blot; motor function by Grip test; brain edema by wet/dry method; cortical apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method; and IL-1ß, TNF-α and IL-6 by enzyme-linked immunosorbent assay (ELISA). We found the absence of TLR2 function in mice resulted in amelio-rating brain injury as shown by the reduced severity of neurological deficit, apoptosis, and brain edema at 24 hours after TBI, which was associated with the decreased expression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), compared with their wild-type counterparts after TBI. In combination, these results suggest that TLR2 might play an important aggravating role in the pathogenesis of TBI-induced secondary brain injury, possibly by regulating inflammatory cytokines in the cortex.

RUNX3 expression is lost in glioma and its restoration causes drastic suppression of tumor invasion and migration.

PURPOSE: The aim of this study is to investigate whether the expression of RUNX3 is related to the development of glioma, and the role of RUNX3 in glioma cells growth, invasion and migration. METHODS: We analyzed the protein expression of RUNX3 by immunohistochemistry in 188 glioma tissues, 8 normal brain tissues and 8 tumor adjacent normal brain tissues using tissue microarray technique. We studied whether RUNX3 restoration can suppress glioma cells growth, invasion and migration by performing MTT cell proliferation assay, matrigel cell invasion assay, wound-healing assay and migration assay. We also detected MMP-2 protein expression and enzyme activity by western blot analysis and gelatin zymography. RESULTS: We found that RUNX3 expression was decreased in benign tumor and malignant tumor compared with tumor adjacent normal brain tissue (P < 0.01 and P < 0.05, respectively). We did not find any correlation between RUNX3 expression and clinicopathological parameters. In addition, we demonstrated that re-expression of RUNX3 in glioma cells resulted in significantly inhibited cell invasion and migration abilities. This reduced cell invasion and migration abilities were due to MMP-2 protein expression and enzyme activity suppression after RUNX3 restoration. CONCLUSIONS: Our data indicated that RUNX3 expression is significantly decreased in human glioma, and targeting of the RUNX3 pathway may constitute a potential treatment modality for glioma.

Dopamine content in the striatum and expression changes of Bad and Bcl-2 in elderly rats with abnormal behavior.

To determine the dopamine (DA) content in the striatum and the expression changes of the apoptosis-associated proteins Bad and Bcl-2 in the substantia nigra compacta (SNc) in elderly rats with abnormal behavior. Fifty three Sprague-Dawley rats were divided into three groups: adult, age-motorplus (normal behavior) and aged-motorminus (abnormal behavior) using the hanger test. The DA content in the striatum and the expression of tyrosine hydroxylase (TH), Bad and Bcl-2 in the SNc were measured by HPLC/MS (high performance liquid chromatogram-mass spectra) and Western Blot. (1) The results from the hanger test demonstrated that the scores and latency of aged-motorminus group were lower than the age-motorplus group. (2) Results from HPLC-MS showed that, compared with the age-motorplus and adult group, the content of DA in elderly rat striata decreased significantly, with a statistically significant difference. (3) The Western Blot demonstrated that, compared with the adults, the expression of TH in elderly rats significantly decreased, but the difference was not significant between the aged-motorminus group and the age-motorplus group. Compared with the age-motorplus and the adult group, the expression of Bad increased but Bcl-2 decreased in the aged-motorminus group. The decrease in TH content in the SNc correlated with the aging of rats. The decrease in DA content in the striatum may correlate with the abnormal behavior in elderly rats, which could be ascribed to the variations in Bad and Bcl-2.

Expressions and possible roles of GDNF receptors in the developing dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) has an essential role in the survival and maturation of the dopaminergic (DA) neurons in the substantia nigra (SN) of mammalian embryonic brain. In addition to Ret, cell adhesion molecules (CAMs) were also proposed to function as transmembrane signaling receptors of GDNF. The present study was to investigate whether these transmembrane receptors of GDNF were correlated with the tyrosine hydroxylase (TH) expression of SN DA neurons during early developmental stage. RT-PCR and Western blot were performed to detect TH expression in SN of perinatal rats at mRNA and protein level respectively; meanwhile, Western blot was performed to detect the expressions of the transmembrane proteins including Ret, neural cell adhesion molecule-140 (NCAM-140), integrin ß1 and N-cadherin. The results showed that TH mRNA expression was positively correlated with both Ret and N-cadherin protein, while there was no correlation with NCAM-140 and integrin ß1; TH protein expression was correlated with all of these transmembrane molecules. These data suggested that the expression of either TH mRNA or TH protein was subject to the mediation of different transmembrane receptor combinations of GDNF.

Effect of intranigral injection of GDNF and EGF on the survival and possible differentiation fate of progenitors and immature neurons in 6-OHDA-lesioned rats.

We investigated the survival and the possible differentiation fate of the progenitors and immature neurons in the pars compacta of the substantia nigra (SNc) by intranigral injection of a glial cell line-derived neurotropic factor (GDNF) or glial cell line-derived neurotropic factor plus epidermal growth factor (EGF + GDNF) in 6-hydroxydopamine (6-OHDA)-lesioned rats. First, we performed behavioral tests by postural asymmetry and forelimb akinesia on the rats injected with 6-OHDA in striatumat day 7, and selected the qualified model according to the results. Then, intranigral GDNF or EGF + GDNF treatment was administered in the qualified PD model rats. On day 21, behavioral tests were performed with these rats; and then the rats were sacrificed for analyses of ß-tubulin isotype-III (Tuj1), nestin, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) by immunohistochemistry and Western blotting. The results indicated that GDNF could promote the survival of the progenitor cells and immature neurons in rat SNc following 6-OHDA lesion. Moreover, EGF is capable of enhancing the survival effect of GDNF on the progenitor cells and immature neurons in SNc. On day 21, rapid functional recovery from the lesion-induced behavioral asymmetries was observed in the GDNF or EGF + GDNF treated rats, and the numbers of TH-positive neurons increased in SNc, suggesting that the rats might generate new dopaminergic neurons. Thus, our study provides the new insight that the progenitors and immature neurons in SNc of 6-OHDA-lesioned rats might be able to differentiate toward the dopaminergic neurons fate subsequent to treatment with GDNF or EGF + GDNF.